运用二代测序技术确认PCR-SSOP法检出的HLA罕见等位基因

目的：确认PCR-序列特异性寡核苷酸探针（SSOP）法检出的4例磁珠探针HLA基因分型结果格局异常和3例模棱两可结果样本的HLA真实基因型。方法：对HLA高分辨组织配型血样采用PCR-SSOP法检出的4例磁珠探针HLA基因分型结果格局异常和3例模棱两可结果进一步采用PCR-直接测序分型（SBT）技术和二代测序（NGS）技术复检确认。结果：采用PCR-SSOP方法检出4例磁珠探针HLA基因分型结果格局异常的样本，加做SBT及NGS两种方法复检后结果显示，样本1的HLA-A分型结果与已知基因型不完全匹配，NGS分析显示，该等位基因与同源性最接近的等位基因A*31:01:02:01在第2外显子154位G>A变异，导致28位编码氨基酸由缬氨酸变为蛋氨酸（p.Val28Met），样本2的HLA-C结果为C*03:119, 06:02，样本3的HLA-C结果为C*03:03, 07:137，样本4的HLA-B结果为B*55:02,55:12。采用PCR-SSOP方法检出3例模棱两可结果样本，加做SBT及NGS两种方法复检后结果显示，样本5的HLA-B结果为B*15:58, 38:02，样本6...     

流式磁珠PCR-SSOP法漏检2例HLA-A位点罕见基因型的原因分析

目的对采用流式磁珠PCR-SSOP法复核2例HLA-A位点罕见基因型,查找分析原因。方法对2 688例HLA-A、B、DRB1SBT基因分型结果中43例罕见型结果,采用流式磁珠PCR-SSOP法复核,查找差异,确定分型,并分析差异结果原因。结果 2例罕见基因型在流式磁珠PCR-SSOP法同一批号03A高分试剂中均有1组磁珠假阳性,造成HLA-A位点罕见基因型漏检。2例罕见基因型在流式磁珠PCR-SSOP法同一批号004高分试剂检测结果与SBT结果一致。结论 HLA罕见基因型不能轻易排除,应用多种方法复核,保证HLA基因分型结果的准确。     

HLA—B27基因定型技术及其临床应用

利用HLA－B27特异性引物扩增其第3个外显子区的DNA片段，建立了HLA－B27的基因定型技术。125例献血员样本和59例临床样本同时用微量淋巴细胞毒试验（血清学方法）和建立的基因定型方法作HLA－B27分型。15株HLA标准细胞用基因定型技术作HLA－B27定型，结果与其真实情况完全相符．基因定型无假阳性和假阴性结果，而血清学方法有2例假阳性。在献血员样本和强直性脊柱炎（AS）样本中，分别有5．6％和91．3％的HLA－B27阳性率，说明B27与AS相关。分析以上结果，可以认为，HLA－B27定型在临床AS诊断中有重要意义，基因定型方法比血清学方法更可靠，也更适合于临床应用。     

中山地区血小板供者HLA基因分型资料库的建立

目的 建立血小板供者HLA基因分型资料库,探讨HLA-A、B基因频率及单体型频率,为临床患者提供HLA匹配的血小板,解决血小板输注无效(PTR)患者的血小板输注.方法 采用聚合酶链反应-序列特异寡核苷酸探针杂交技术 (PCR-SSOP)对568名中山地区单采血小板捐献者进行HLA-I类(A、B位点)基因分型并形成数据库,根据HLA表型群体资料,使用最大数学预期值算法(EM)计算HLA-A、B单体型频率.结果 HLA-A位点共检出17种等位基因,HLA-B位点共检出29种等位基因,单体型数153种.结论 血小板供者HLA分型资料库的建立可以为PTR患者迅速寻找到HLA相合血小板,提高血小板输注效果.     

山东省汉族人群HLA-A、B、DRB1位点基因多态性研究

目的分析山东省汉族人群HLA-A、B、DRB1位点基因多态性,获得更完整、准确的群体HLA分子遗传学数据。方法应用PCR-SSP及SSOP技术对7418名山东省汉族造血干细胞志愿捐献者进行HLA-A、B、DRB1位点基因分型。结果共检出低分辨HLA-A基因18种,B基因44种,DRB1基因13种,其中包括一些以前国内未检出的低频率基因。山东汉族人群最常见的HLA基因为A*02、B*13和DRB1*15,其相应基因频率分别为0.2883、0.1431和0.1762;山东汉族人群最常见的A-B单倍型为A*30-B*13,频率为0.0896,最常见的A-B-DRB1单倍型是A*30-B*13-DRB1*07,频率为0.0743。结论山东省汉族人群HLA-A、B、DRB1基因具有显著多态性,大样本和DNA分型有助于低频率基因的检出,适当扩大造血干细胞库志愿捐献者登记人数及各地重新进行HLA多态性分析实有必要。     

应用PCR—SSP进行HLA—AB基因分型及与血清学方法的比较

目的 建立HLAⅠ类基因分型技术并应用于骨髓七配型。方法 采用序列特异性聚合酶链反应（PCR－SSP）进行HLA－AB基因分型，并与HLAⅠ类血清学分型（微量淋巴细胞毒试验）进行比较。结果 运用PCR－SSP技术共对22例需骨髓移植的病人和48例供者进行基因分型，2例病人确认了HLA 新缘骨髓供者，其中1 已成功进行了骨髓移植；基因分型和血清分型比较，43例结果完全一致（61.4%），血清分型错误率高达38.6%(27/70)。结论 PCR－SSP分型技术具有准确性高，简便快速等优点，将取代传统的血清学方法。     

强直性脊柱炎与HLA—B27

强直性脊柱炎是HLA关联最强的疾病。HLA－B27由22个以上同种异型基因型（亚型：B*2701-B*22)组成，不同亚型核苷酸序列之间只存在个别位点的差异，其亚型具有分布不同的种族和人种充行情况，以B*2705分布最广。近年来建立了大量的AS动物模型，人类B27转基因鼠实验证实B27分子是AS的原发关联成分。PCR－SSP等DNA分型技术和流式细胞术克服了传统的血清学分型技术检测HLA－B27的众多弊端，成为临床辅助诊断强直性脊柱炎的重要手段。     

脐血HLA-AB分型方法探讨

目的 探讨脐血HLA-AB分型方法。方法 分别采用血清学方法和PCR-SSP基因定型方法对102份脐血HLA-A、B位点进行检测。结果 两方法对比分析结果显示血清学近18.6%不能得出满意结果，HLA-A、B位点错定率分别为6.1%和10.8%，总错误率16.9%。结论 脐血HLA表达水平低，单个核细胞反应弱及部分交叉反应是造成血清学不能确定或错定的主要原因。     

新等位基因HLA-A~*1127的确认和序列分析

目的识别确认临床样本中一个新的HLA等位基因。方法应用PCR-SSO,PCR-SSP基因分型技术对HLA分型,对可能的HLA新等位基因进行分子克隆和DNA测序,分析与最同源的A*1104的差异。结果该序列与已知的所有HLA-A等位基因序列不一致,与A*1104的差异表现在第3外显子区域中的核苷酸570 G→C,571 T→G导致氨基酸分别由谷氨酸→天冬氨酸,色氨酸→甘氨酸。结论该基因为HLA-A位点的一个新等位基因,已被WHO HLA因子命名委员会于2007年8月正式命名为HLA-A*1127。     

江苏骨髓分库汉族造血干细胞捐献志愿者HLA-A、B、DRB1基因多态性和单倍型研究

目的分析中国造血干细胞捐献者资料库(简称江苏分库)汉族人群中HLA-A,B,DRB1基因多态性和单倍型的分布特征。方法用PCR-SSP和PCR-SSOP基因分型技术对江苏分库20 248名无关志愿者作HLA-A、B、DRB1低分辨基因分型,以直接计数法计算等位基因频率,应用Arlequin 3.01软件以最大似然法分析单倍型频率。结果江苏分库汉族志愿者HLA抗原频率分布符合Hardy-Weinberg平衡;共检出HLA-A位点等位基因18个,B位点等位基因34个,DRB1位点等位基因13个;A位点频率最高的是A*02(29.6%),B位点频率最高的是B*15(14.4%),DRB1位点频率最高的是DRB1*09(16.2%);频率最高的HLA-A,-B,-DRB1单倍型是A*30-B*13-DRB1*07(6.92%),频率最高的HLA-A、B单倍型是A*30-B*13(8.04%),频率最高的HLA-B,DRB1单倍型是B*13-DRB1*07(8.07%),频率最高的HLA-A、DRB1单倍型是A*02-DRB1*09(7.70%)。结论对江苏分库汉族人群HLA的分布状况的了解,有助于指导临床寻找HLA匹配的无关骨髓供者,为HLA与疾病相关研究和我国人群群体遗传学研究等提供了有意义的基础性资料。     

西安地区HLA-B*53频率及高分辨确认分析

目的调查西安地区HLA-B*53基因频率并进行高分辨确认。方法采用序列特异性引物(PCR-SSP)和特异性寡核苷酸探针(PCR-SSO)方法对HLA-B*53进行低/中分辨调查和高分辨确认。结果13 945样本中有13例样本低分辨认为可能是HLA-B*53,其中6例样本经高分辨确认为HLA-B*5301,另有7例样本被证实不是B*53。结论西安地区HLA-B*53基因频率为0.0002,在遇到模棱两可的分型结果时,要采用多种检测方法综合分析,确保HLA分型结果的准确性。     

DNA序列分析鉴定HLA新等位基因B~*0740

目的确认一个中国人群中的新HLA等位基因。方法应用单倍型特异性分离(haplotype-specific ex-traction,HSE)技术和DNA序列分析技术鉴定HLA-B~*的新等位基因。结果被检标本HLA-B位点有一个等位基因的核苷酸序列与任何已知的HLA等位基因均不同,与同源性最高的B~*0705相比,在exon3区域中的605位碱基由A>T,引起编码178位的氨基酸由赖氨酸(K)变成甲硫氨酸(M)。血清学分型表明新等位基因的免疫学表型仍为HLA-B7。家系分析提示,HLA-B~*0740基因来源于其母亲。结论该等位基因序列为HLA-B位点的一个新等位基因,于2005年2月由WHO HLA因子命名委员会正式命名为HLA-B~*0740。     

Incidence of abacavir hypersensitivity and its relationship with HLA-B*5701 in HIV-infected patients in Taiwan

OBJECTIVES: To describe the incidence of hypersensitivity to abacavir and frequency of human leucocyte antigen (HLA)-B*5701 in HIV-infected Taiwanese persons. METHODS: Medical records of 337 HIV-infected Taiwanese in whom abacavir-containing combination antiretroviral therapy (CART) was prescribed from 1 May 2001 to 31 December 2006 were reviewed, and HLA typing of the patients was performed in 320 patients (232 receiving abacavir and 88 not receiving abacavir) with available blood samples. HLA class I and II polymorphisms were determined by PCR with specific primers. HLA-B*5701 was further confirmed by sequence-based typing. RESULTS: Of the 337 patients, median CD4 count was 166.5 cells/mm3 (range, 1.0-1914.0) and 83 patients (24.6%) had AIDS-defining opportunistic infections. Thirty-eight patients (11.3%) discontinued abacavir within 6 weeks of starting abacavir-containing CART. Among them, 10 patients had successful abacavir re-challenge and another 11 patients had other specific reasons for abacavir discontinuation. Therefore, 14 patients (4.2%) were classified as cases in whom abacavir hypersensitivity could not be excluded, and 3 patients (0.9%) met the criteria of abacavir hypersensitivity. Of the 320 patients undergoing HLA typing, HLA-A02 was the most common allele and only one individual (0.3%) expressed HLA-B*5701. Along with some differences in allele distributions, there was a significant difference in the genetic frequency of HLA-B57 in our patients compared with those of previous studies in other Chinese populations. CONCLUSIONS: Abacavir hypersensitivity was less frequently encountered in HIV-infected Taiwanese initiating abacavir-containing CART than in Caucasians, which might be explained by the low frequency of the HLA-B*5701 allele.     

External quality assessment of HLA-B*5701 reporting: an international multicentre survey

OBJECTIVES: HLA-B*5701 strongly predicts abacavir hypersensitivity (HSR), but implementation of effective routine screening into clinical practice requires testing be practical and accurate. We tested the proficiency of HLA-B*5701 typing among laboratories using sequence-specific primer PCR. DESIGN AND METHODS: DNA panels (1 and 2) were distributed to seven laboratories (A to G) for blinded typing of the HLA-B*5701 allele. Panel 1 (n = 10 samples; n = 7 laboratories) included 3 positives and other closely related B17 subtypes (B*5702, B*5703, B*5704 and B*5801). Panel 2 (n = 96 samples; n = 4 laboratories) included 36 positives among a broad spectrum of other B alleles. Two laboratories (A and B) also submitted 96 routine samples, typed by the same methodology, to the reference centre for additional analysis by sequence-based typing. RESULTS: All laboratories correctly typed panel 1 for HLA-B*5701 carriage. Laboratories A, B and C identified HLA-B*5701 alleles in panel 2 with 100% sensitivity and 100% specificity. Laboratory D reported one false negative, reportedly due to a sampling error. The results obtained for routine samples typed by laboratories A and B and those generated by the reference laboratory using sequencing were fully concordant. CONCLUSIONS: Detection of HLA-B*5701 alleles among laboratories was 100% specific and 99.4% sensitive, indicating that participating HIV testing laboratories were currently offering effective primary screening to identify individuals at high risk of abacavir HSR. Accurate reporting of HLA-B*5701 status is critical for the safe administration of this drug and participation in quality assurance programmes by all sites who report HLA-B*5701 status should be promoted.     

标准血清学法,单克隆抗体法和 NA法行HLA—A抗原分型的比较

目的：双盲比较研究血清学方法、单克隆抗体（单抗）方法和ＤＮＡ方法用于中国汉族人群ＨＬＡ－Ａ抗原分型的精确性。方法：研究样本２９６份,包括１４３名无关供者和１５３例等待肾移植受者。血清学方法为二步法微量淋巴细胞毒分型技术;单抗法为一步法单抗分型技术;ＤＮＡ方法采用顺序特异引物聚合酶反应（ＰＣＲ－ＳＳＰ）技术。结果：所有样本ＤＮＡ分型匀获得成功,重复率１００％,分型结果经标准ＤＮＡ验证和美国加州大学配     

A new approach to safely type for HLA the HIV infected people eligible to abacavir therapy: Saliva or buccal swab as reliable DNA sources

BackgroundIncreasing the safety in Immunogenetics Labs, in the era of antiretroviral pharmacogenomics, represents an imperative goal. To this purpose, we tested saliva and buccal cells as biological sources of DNA, alternative to peripheral blood, for HLA-B*57:01 genomic typing of HIV positive patients eligible to treatment with abacavir.MethodsBlood, saliva and buccal cells of 20 voluntary donors and 20 HIV positive patients were collected. DNA was extracted with a manual commercial kit and an automated platform. Quality and quantity of DNA was evaluated with different procedures. The suitability and reliability of DNAs for HLA-B*57:01 genotyping was checked at low and high resolution level, using PCR-SSP (sequence specific primers PCR), revPCR-SSO (reverse sequence specific oligonucleotides PCR), bead array and SBT (sequence based typing) techniques.ResultsDNA concentrations were qualitatively very good and quantitatively comparable in all the specimens tested with an inferior yield for cotton swabs. Comparing the results of HLA typing with different methodologies, the 100% of reproducibility was achieved.ConclusionsThe viral load of buccal epithelial cells or saliva is extremely low. Here we demonstrated that the DNA from these alternative sources is appropriate for HLA-B*57:01 typing. We strongly recommend the use of this procedure to increase the safety in the lab when dealing with infectious samples.     

山东半岛地区汉族人群HLA-A、B、DRB1高分辨等位基因及单体型多态性的研究

目的研究山东半岛地区汉族人群HLA-A、B、DRB1等位基因多态性的分布特征。方法应用聚合酶链反应-直接测序分型(polymerase chain reaction sequence-based typing,PCR-SBT)法和序列特异性寡核苷酸探针杂交技术(Polymerase chain reaction and sequence-specific olignucleotide probe hybridizations,PCR-SSOP)高分辨试剂,对山东半岛地区865名无血缘关系的汉族健康人群HLA-A、B、DRB1进行基因分型。结果检出HLA-A、B、DRB1等位基因分别有33、73、43种,经统计分析这3个基因座分布均符合Hardy-Weinberg平衡定律(P>0.05),A*3001-B*1302-DRB1*0701(7.61%)和A*3303-B*4403-DRB1*1302(1.674%)单体型是山东半岛地区汉族人群最常见单体型。结论山东半岛地区汉族人群HLA-A、B、DRB1基因座单体型分布具有高度的遗传多态性且有其自身分布特点。本研究获得的的HLA-A、B、DRB1基因座单体型分布数据及相关遗传参数资料,为HLA在人类学、免疫遗传学、法医学和其它的组织器官移植方面的应用和科学研究提供和积累了基础资料。